Studies on the incorporation of U-14C-glucose
into vitreous polymers in vitro and in vivo
E. R. Berman and G. M. Gombos
The incorporation of U-^C-glucose into the nondialyzable polymers of the vitreous was
studied both in vitro, xoith bovine tissue preparations, and in vivo, after the infusion of
radioactive glucose through the lateral long ciliary artery of the cat. The radioactivity present
in glucosamine isolated after Doioex 50 chromatography was taken as a measure of hyaluronic
acid synthesis. It was found that cortical layer vitreous from young (4-week-old) calves was
more active in converting glucose to glucosamine than equivalent samples taken from adult
animals. No striking differences in biosynthetic activity toere observed between the vitreous
alone or with attached ciliary body, suggesting that under these experimental conditions the
ciliary body does not contribute to the production of vitreous hyaluronic acid. The central
(acellular) regions of the vitreous toere nearly as active as the peripheral regions in incorporating labeled glucose into nondialyzable polymers, a finding which could be ascribed to
"extracellular enzymes" in the vitreous. Studies of the distribution of radioactivity in purified
fractions shotoed. that most of the glucose had been utilized for the synthesis of proteins
(and glycoproteins). Incorporation of radioactive glucose into nondialyzable polymers of the
vitreous was inhibited by approximately 80 per cent in the presence of puromycin, due
mainly to the depression of protein and glycoprotein synthesis. Very little incorporation of
U-'J'C-glucose into hyaluronic acid occurred in vivo, although under the same experimental
conditions appreciable amounts of radioactivity were found in the amino sugar moieties of
the corneal mucopolysaccharides.
Key words: vitreous, glucose, glucosamine, hyaluronic acid, protein synthesis,
glycoproteins, mucopolysaccharides, cornea, puromycin, pars plana, cats.
occur in it during lifetime (and especially in senescence) appear to be essentially
irreversible. Nevertheless, our knowledge
of its metabolic patterns is very meager;
the biosynthesis of its macro molecular components appears to be limited in nature
and occurs mainly during periods of active growth and development. These and
other aspects of vitreous chemistry and
metabolism have been recently reviewed.1
Both synthetic-- b and hydrolytic'1 enzymes have been detected in high-speed
sediments of vitreous pooled from approximately 100 calf eyes. The latter enzymes
are localized mainly in the "granular"
fraction of the vitreous cells (hyalocytes)
.he metabolism of the vitreous body
is of particular interest not only because
the tissue occupies a major portion of the
globe in nearly all vertebrate species but
also because the degenerative changes that
From the Department of Ophthalmology, Biochemistry Research Laboratory (The Sir Isaac
and Lady Wolfson Ophthalmic Research Laboratories), Hebrew University-Hadassah Medical School, Jerusalem, Israel.
These investigations were supported in part by
the United States Public Health Service, National Institutes of Health Grant NB-05887, and
the Michael Polak Research Fellowships in
Ophthalmology.
521
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Investigatioe Ophthalmology
October 1969
522 Berman and Gombos
and resemble in several respects lysosomal
enzymes isolated from other tissues. The
histochemical and morphological properties of the cortical layer cells have been
studied extensively5"8 and it has been proposed that they play an important role in
the biosynthesis of vitreous hyaluronic
acid.3j ° That the ciliary body may also
take part in its production may be inferred
from histochemical studies in the developing rabbit eye.10 Light and electron microscopic evidence suggests that the acid
mucopolysaccharides found in the intercellular spaces of the nonpigmented epithelium of the ciliary body may be transported into adjacent areas of the vitreous
through narrow channels or "intercellular
canaliculi."11 This hypothesis was strengthened by the observation that hyaluronidase-sensitive mucopolysaccharides not only
accumulate within cysts and neoplastic
growths of the ciliary epithelium but also
are extruded into the surrounding extracellular spaces.12
In mammalian tissues and in bacteria13
both the glucuronic acid and the glucosamine moieties of hyaluronic acid are derived directly from glucose. The assumption that a similar pathway exists in the
vitreous is based mainly on a report9 describing the incorporation of glucose-14C
into hyaluronic acid in vitro after incubation of portions of cortical tissue layer.
Nevertheless, parenterally administered
glucose-14C is not incorporated into the
hyaluronic acid of owl monkey vitreous,3
nor does it appear to be an effective precursor of any nondialyzable macromolecular components in the cat vitreous.14
The present investigations were prompted
mainly by the scarcity of precise information on the extent to which (radioactive)
glucose is utilized for hyaluronic acid
synthesis by the vitreous body. In addition, attempts have been made to establish the principal site(s) of metabolic activity in the vitreous itself and to determine whether the ciliary body participates
in the production of vitreous hyaluronic
acid.
Materials and methods
In vitro experiments. Cattle eyes from the following three age groups were used: (1) 4-weekold calves, (2) 3- to 6-month-old calves, and (3)
1- to 2-year-old cattle. They were brought from
the slaughterhouse on ice and dissected immediately. After die cornea, iris, and lens were removed, the exposed vitreous was cut circularly
at a point 3 to 4 mm. from the peripheral edge
of the tissue to a depth of approximately 5 mm.
In this way it was possible to separate the vitreous lying directly adjacent to die ciliary body
from the rest of the vitreous tissue. An incision
5 mm. deep was then made (posteriorly) across
the ciliary body and sclera, and the tissues were
then cut around the entire circumference. The
resulting strip of tissue, measuring 7 to 10 cm. in
length (depending on the age of the animal),
was held firmly at one end with a small forceps
and the ciliary body (together with the vitreous
adhering to it) was gently pulled away from the
sclera. This preparation, consisting of intact ciliary
body togedier with 2 to 3 ml. of cortical layer
(pars plana) vitreous, has been designated Preparation A. Four identical strips were used for each
experiment. Another set of four cattle eyes was
dissected in the same manner except that the
ciliary body-vitreous preparation was pinned to
a small cork board and the vitreous was carefully
removed. This vitreous tissue, originating mainly
in the pars plana region, has been designated
Preparation B. Preparation C consisted of equivalent amounts of vitreous gel taken only from
the central region of the tissue. These preparations were then incubated with U-^'C-glucose
(Amersham, England) for 6 hours unless otherwise indicated.
In vivo experiments. Cats weighing 2Yz to 3
kilograms were used in all experiments. The
U-14C-glucose (100 /ic), dissolved in 6.5 ml. of
0.9 per cent NaCl, was infused through the lateral long ciliary artery34 for 4 hours at a rate of
1.6 ml. per hour. At the end of the experiment
both the cornea and the vitreous were removed
and frozen at -25° C. Five identical experiments
were carried out and the vitreous and corneas
from each were then pooled.
Measurment of total radioactivity incorporated.
In vitro experiments. After incubation of the
vitreous Preparations A, B, and C, each was centrifuged at 35,000 x g for 1 hour and dialyzed
overnight in a Perspex cylinder against 5 to 6
L. of continously running deionized water. Control experiments with the use of approximately
the same amount of radioactivity had shown that
this method was effective in removing all of the
free (nonincorporated) radioactive glucose. Therefore, the amount of radioactivity remaining after
dialysis under these conditions was considered a
valid estimation of the extent of incorporation into
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Volume 8
Number 5
U-'J>C'-glucose and vitreous polymers
nondialyzable polymers. All samples were counted
in a Packard Tri-Carb liquid scintillation spectrometer as described previously.15
In vivo experiments. The pooled preparations
obtained from the infusion experiments were
treated as follows. Ten "carrier" corneas from
(enucleated) cats' eyes were added to the corneas
from the five perfused eyes and all of them
extracted twice with water and twice with 0.1M
K.CO-i in a VirTis homogenizer. The extracts were
combined, centrifuged at 37,000 x g for 2 hours,
and then dialyzed exhaustively in a Perspex cylinder. Radioactivity was measured as described
for the in vitro preparations.
"Carrier" vitreous from 10 cats' eyes was added
to the vitreous from the five perfused eyes. After
centrifugation at 35,000 x g for 1 hour, the
supernatant was dialyzed exhaustively and
counted.
Ethanol precipitation. Hyaluronic acid was
precipitated from the dialyzed vitreous samples
by the addition of 3 volumes of ethanol in the
presence of sodium acetate (25 mg. per milliliter). After standing overnight at 4° C. the solution was centrifuged at 20,000 x g for 20 minutes
and the residue taken up in water. Insoluble
materials (mainly denatured proteins) were removed by centrifugation and the hyaluronic acid
was again precipitated with ethanol, dissolved in
water, and dialyzed.
Separation of hyaluronic acid and proteins.
Vitreous preparations that had been incubated
with radioactive glucose were centrifuged and
dialyzed, first against water (as described above)
and then against 0.005M phosphate buffer, pH 7.
The sample was applied to a column (1 by 10
cm.) of DEAE-Sephadex that had previously
been equilibrated with the same buffer; the fractions were eluted in a closed system gradient of
Separation of mucopolysaccharides and pep'
tides. Centrifuged vitreous or corneal extracts
were incubated for 16 hours at 37° C. with
Pronase (Calbiochem AC, Luzern, Switzerland)
in the presence of 2 to 3 drops of toluene, using
1 A<g of enzyme for each 50 /*g of protein. The
digested preparations were concentrated to a
volume of 2 to 3 ml. and chromatographed on
columns of Sephadex G-25 (2.5 by 40 cm.)
previously equilibrated with 0.02M phosphate
buffer, pH 7. The fractions were eluted in the
same buffer at a flow rate of 20 ml. per hour.
Isolation of aniino sugars. Samples were hydrolyzed in 2N HC1 for 12 hours at 100° C. and
the HC1 was evaporated in vacuo over P2O5 and
NaOH. The dried samples were dissolved in 0.3N
HCl and chromatographed on columns (1 by 25
cm.) of Dowex 50 according to Gardell.18
Chemical analyses. Uronic acid and protein
were measured as described previously.35 Hexosamine was assayed by a modification19 of Elson
and Morgan's20 procedure and neutral sugar by
a modification of the anthrone reaction.21
523
Results
In vitro experiments.
Incorporation into glucosamine. The experimental conditions leading to maximum
incorporation of radioactive glucose were
determined by examining the influence of
(1) the amount of radioactivity in the
incubation medium, (2) the age of the
animal, and (3) the length of time of the
incubation. Moreover, in order to gain
some information on the principal biosynthetic site(s) in the vitreous and also to
assess the role of the ciliary body in the
Table I. Incorporation of IP'C-glucose into nondialyzable polymers of calf vitreous'
Preparation
Vitreous (ciliary) A
Vitreous B
Vitreous C
Initial
radioactivity
(nc)
10
10
10
Uronic
acid
(W)
746
800
1,100
Protein
(mg.)
36.2
6.2
3.8
Radioactivity
incorporated
(counts/min.)
19,800
17,400
14,160
Vitreous (ciliary) A
Vitreous B
Vitreous C
20
20
20
1,040
975
1,520
49.4
3.8
4.1
18,000
25,000
15,000
Vitreous (ciliary) A
Vitreous B
Vitreous C
50
50
50
694
782
1,020
33.2
5.4
3.4
56,000
52,700
37,500
"The vitreous samples were prepared from 3- to 6-month-old calf eyes as described in Materials and methods. Each
flask contained approximately 10 ml. of the designated preparation plus glutamine (1 mg. per milliliter) and unlabeled
glucose (0.5 mg. per milliliter). Incubations were carried out in an atmosphere of 95 per cent oxygen and 5 per cent
CO2 for 6 hours at 37° C.
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Investigative Ophthalmology
October 1969
524 Berman and Gombos
Table II. Distribution of radioactivity after ethanol precipitation and Dowex 50 chromatography*
Glucosamine
Preparation
Beforef
630
710
984
After
405
493
582
Ethanol precipitation
Protein
(mg.)
Before
After
33.2
8.8
5.4
1.2
3.4
0.9
Dowex 50
chromatography
Radioactivity
(counts/min.)
Before
After
55,000
51,500
33,500
9,700
6,100
4,400
Glucosamine
(»g)
265
350
390
Radioactivity
(counts/
min.)
80
105
95
•Vitreous-ciliary preparations from 3- to 6-month-old calf eyes were incubated for 6 hours at 37° C. with 50 /ic of
U-KC-ulucose. Other conditions were the same as those described in Table I.
f Before and after ethanol precipitation.
production of hyaluronic acid, three different tissue preparations were used (see
Materials and methods).
The results of experiments with 3- to
6-month-old calf eyes are summarized in
Table I. Although increasing the amount
of radioactive glucose in the medium from
10 to 20 /.ic did not result in a corresponding increase in radioactivity incorporated,
50 /.ic of labeled glucose caused a substantial rise. However, regardless of the amount
of radioactivity initially present, there was
no clear indication of where the principal
biosynthetic sites were located. In most
cases it appeared that the central (acellular) portion of the vitreous (Preparation
C) was somewhat less active than the pars
plana region (Preparation B), which has
the highest concentration of vitreous cells.7
However, the finding that so much radioactivity was incorporated by the acellular
portions at all was unexpected since most
of the metabolic activity of the tissue is
believed to reside in the cortical layer
cells.2"1'9 It should also be noted (Table
I) that there was relatively little difference
in the amount of labeled glucose incorporated by Preparation A (pars plana vitreous plus ciliary body) and Preparation
B (pars plana vitreous alone), implying
that the ciliary body, under these experimental conditions, does not play an active
role in the biosynthesis of the nondialyzable vitreous polymers.
The data given in Table I represent the
total radioactivity incorporated into both
hyaluronic acid and proteins, the principal
soluble nondialyzable polymers of the
vitreous. The amount specifically incorporated into the hyaluronic acid in the six
vitreous samples that had been incubated
with either 10 or 20 [xc of labeled glucose
was determined after Dowex 50 chromatography. Even though the amino sugars
were recovered in yields of 60 to 70 per
cent, no radioactivity could be detected
in any of the six samples.
Examination of the samples incubated
with 50 ju.c of U-14C-glucose showed that
after ethanol precipitation there was a
marked shift in the distribution of radioactivity (Table II). Although 60 to 70 per
cent of the hyaluronic acid was recovered,
only about 10 to 20 per cent of the radioactivity was associated with it. The rest
of the radioactivity was lost, together with
approximately 75 per cent of the protein.
When these samples were hydrolyzed and
chromatographed on Dowex 50, the glucosamine isolated contained approximately
100 c.p.m., representing 1 to 2 per cent
of the total counts applied to the Dowex
50 column.
Thus, although very little glucose had
been incorporated into the hyaluronic acid
under the experimental conditions described
above, it was still necessary to determine
whether the age of the animal or the length
of time of the incubation could influence
the extent of conversion to glucosamine.
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U-'J'C-glucose and vitreous polymers 525
Volume 8
Number 5
Table III. Effect of age and time of incubation on the incorporation of U-niC-grucose into
vitreous hyaluronic acid*
Tissue preparation
Time of
incubation
(hr.)
U ionic
acid
Radioactivity (counts/min.)
In nondialyzable
polymers
In glucosamine
after Dowex 50
4-week-okl calves
6
30f
610
680
98,150
90,400
550
6S0
3- to 6-month-okl calves
6
30 f
830
740
56,700
43,100
SO
108
1,020
30,200
140
6
86
950
38,800
86
950
30J
30f
"Each incubation flask contained 50 /ic of U-lt!C-glucose in a total volume of approximately 10 ml. In the experiments
with 3- to 6-month-old calves and 1- to 2-year-old cattle, vitreous with attached ciliary body (preparation A) from
of hyaluronic
4 ey
eyes was used;; but
but for
for 4-week-okl
4-week-okl calves,
calves, 8
8 eyes
eyes were
were required
required in
in order
order to
to obtain
obtain equivalent
equivalent amounts
amounts of
acid.
acid,
fPenicillin (100 units per milliliter) and streptomycin (50 tig per milliliter) were added to these preparations.
1- to 2-year-old cattle
It has been shown by Balazs and co-workers7' — that not only is the increase in the
concentration of hyaluronic acid greatest
in the early postnatal period of growth22
but also the number of cells in the peripheral layer of the tissue (especially in
the pars plana region) is higher in calves
than in adult cattle.7 Preparation A (pars
plana vitreous with attached ciliary body)
was used in all subsequent in vitro experiments even though the data in Tables
I and II do not support the view that
the ciliary body plays a direct role in
the biosynthesis of vitreous hyaluronic acid.
However, it may be assumed that in Preparation A little or no damage to the vitreous cells had taken place, whereas in Preparation B, which requires stripping the
vitreous from the ciliary body, some damage or loss of cells may have occurred.
The results of these experiments (Table
III) show that, although the time of incubation does not appear to be an important
factor, the age of the animal is. The
radioactivity incorporated was 2 to 3 times
greater for 4-week-old calves than for
older animals. Differences in biosynthetic
activity between the age groups are even
more marked when considering the synthesis of radioactive glucosamine (Table
III, last column). In this case there was
5 to 6 times more radioactivity in the
amino sugar isolated from 4-week-old calf
vitreous than from older animals.
DEAE-Sephadex chromatography. Indirect evidence from the above experiments suggests that most of the incorporated radioactivity is associated with the
proteins (and glycoproteins). In order to
assess this more accurately, the experiments
were repeated and the dialyzed samples
chromatographed on DEAE-Sephadex. As
shown in Fig. 1, two well-defined peaks
appear in the elution profile, the first one
consisting of proteins (and glycoproteins)
and the second one of hyaluronic acid. Almost all of the radioactivity was eluted in
the first peak, whereas only negligible
amounts were found in the later (hylauronic acid) fractions. In similar experiments
on 4-week-old calf vitreous, although more
radioactivity appeared in the second (hyaluronic acid) peak, the greater part of it
was still found in the protein and glycoprotein fractions.
Gel filtration and puromycin. Further
experiments to determine the extent of
conversion of labeled glucose to protein
components were carried out using calf
eyes from both 4-week-old and 3- to 6month-old animals, the age depending
mainly on the availability of material at
the slaughterhouse. After incubation with
50 i-i.c of labeled glucose, the preparations
were dialyzed, digested with Promise, and
applied to columns of Sephadex G-25. In
the gel filtration diagram shown in Fig.
2, hyaluronic acid is eluted first as a single
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Investigative Ophthalmology
October 1969
526 Berman and Gombos
o
-1000
1.0 - — 100
00
Cvl
5,
g
LU
0.5
<
- 500 >
50
CD
u
cr
o
o
cr
CO
m
<
0
20
TUBE
cr
30
No-
Fig. 1. Fractionation of cattle vitreous on DEAE-Sephadex after incubation with U-14C-glucose. The effluent fractions were analyzed for protein (•
• ) , uronic acid (x- -x), and
radioactivity (O
O).
-.400
1.5
I
300
75i-
200
1.0
UJ
>
2
U
o
GO
O
CO
100
0.5
Q
a:
m
<
150
EFFLUENT
200
250
Fig. 2. Gel filtration on Sephadex G-25 of Pronase-digested cattle vitreous after incubation
14
• ) , uronic
with U C-glucose. The effluent fractions were analyzed for ninhydrin ( •
O) and radioactivity (x
x).
acid (O
symmetrical peak with the void volume of
the column. This fraction (I) also contained
some ninhydrin- and anthrone-positive material, probably representing either high
molecular weight glycopeptides or Pronase-resistant glycoproteins.23'25
Approximately one quarter of the total
radioactivity eluted from the column was
present in Fraction I. It should be noted,
however, that the principal radioactive peak
did not coincide with either the hyaluronic
acid or the low molecular weight peptides
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U-UC-glucose and vitreous polymers 527
Volume 8
Number 5
2.0 r-
100 r—
300
1.5
1.0 -
r^ 50
200
-
CD
Q:
o
tn
m
<
100
0.5
a
<
Q:
0
L-
50
100
ml
150
250
EFFLUENT
Fig. 3. Gel filtration of puromycin-treated vitreous preparation after incubation with U-14Gglucose and Pronase digestion. The effluent fractions were analyzed for ninhydrin ( •
•),
uronic acide (O
O) and radioactivity (x
x).
and amino acids, which were eluted at the
end of the chroinatogram. The fractions
were pooled as indicated in Fig. 2 and
analyzed together with the puromycintreated preparations described below.
Parallel experiments were carried out
under identical conditions except that,
prior to adding the radioactive glucose,
the vitreous was preincubated with puromycin (100 ^g per milliliter) for 15 minutes. Gel filtration of a puromycin-treated
Pronase-digested preparation (Fig. 3)
showed the same elution pattern for the
hyaluronic acid and the ninhydrin-positive
substances, but revealed striking quantitative differences in the radioactive peaks,
both of which were markedly depressed.
Inhibition of protein synthesis by this antibiotic is well known and similar effects
on the biosynthesis of mucoproteins-0 and
of galactosamine-containing proteinpolysaccharides27"29 have also been demonstrated.
Hyaluronic acid represents a special case
since the biosynthesis of this polysaccharide is less sensitive to puromycin than other substances,20 possibly
because of its lower protein content.
Hydrolysis and (Dowex 50) chromatography of Fractions I and II obtained after
gel filtration of the Pronase digests showed
that two thirds of the total radioactivity
recovered was present in the ninhydrinpositive fractions (Table IV). In absolute
terms, the conversion of labeled glucose
to amino acids and to neutral sugar was
strongly inhibited (71 to 85 per cent) in
the presence of puromycin, whereas conversion to glucosamine was depressed by
only 28 per cent. The over-all inhibition
by puromycin (Table IV, last column),
therefore, reflects mainly a decrease in
the conversion of labeled glucose to amino
acids and neutral sugar (mainly galactose),
thus providing further evidence that the
glucose is being utilized mainly for the
synthesis of proteins and glycoproteins and
to only a limited extent for the synthesis
of hyaluronic acid.
In vivo experiments. Previous investigations1* had shown that although considerable radioactivity entered the vitreous body
after the infusion of labeled glucose
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Investigative Ophthalmology
October 1969
528 Berman and Gombos
Table IV. Effect of puromycin on the distribution of radioactivity in fractions* isolated
after gel filtration and Dowex 50 chromatography
Fraction II
Sample
Control
Puromycintreated
Per cent
inhibition
Fraction I
Neutral sugar
Glucosamine
RadioRadioAmount
activity
Amount
activity
(c.p.m.)
(c.p.m.)
(rg)
132
113
785
87
73
120
102
85
95
Ninhudr inpositive
fractions
(counts/
min.)
2,258
i
Neutral sugar
Radioactivity
Amount
(c.p.m.)
.
J
Total
radioactivity
recovered
(counts/
min.)
3,360
28
185
32
55
515
785
71
78
77
28
0
After incubation with U-^C-glucose, the vitreous samples were digested with Pronase. Two principal fractions (I and
II) were then isolated after Sephadex G-25 chromatography (see Figs. 2 and 3). Each was hydrolyzed and chromatographed on Dowex 50. The anthrone-positive fractions (neutral sugar) appear in the first Dowex 50 effluents. The
glucosamine in Fraction I and the ninhydrin-positive substances (peptides and amino acids) in Fraction II appear in
the effluent fractions eluted afterward with HC1.
CORNEA
IN VIVO
300
1.5 I-
250
200
150 -_
t—
>
100 O
o
Q
50 %
ml EFFLUENT
Fig. 4. Gel filtration on Sephadex G-25 of Pronase-digested corneal extract after infusion
of U-14C-glucose through the lateral long ciliary artery of the cat. The effluent fractions
were analyzed for ninhydrin ( •
• ) , neutral suger (O
O), uronic acid (A
A)
and radioactivity (x
x).
through the lateral long ciliary artery of
the cat, very little remained after dialysis.
It has subsequently been found that in order to study the in vivo metabolism of
labeled glucose in greater detail, larger
amounts of radioactive glucose had to be
administered. Moreover, it was necessary
to pool the vitreous from 5 separate experiments and to add "carrier" vitreous
so that sufficient hyaluronic acid would
be available for subsequent purification.
As a check on the in vivo technique it-
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U-U'C-glucose and vitreous polymers 529
Volume 8
Number 5
1.0 h
VITREOUS
200 h-
o
in
I
LU 0.5
o
Z
CD
QC
O
if)
CO
50
75
100
125
150
200
ml EFFLUENT
Fig. 5. Gel filtration on Sephadex G-25 of Promise-digested vitreous after infusion of U-14Cglucose through the lateral long ciliary artery of the cat. The effluent fractions were analyzed
for ninhydrin ( •
• ) , uronic acid (O
O) and radioactivity (x
x).
self, the incorporation of glucose into the
mucopolysaccharides of the cornea under
the same experimental conditions was also
examined.
After infusion of the radioactive glucose
and exhaustive dialysis of the pooled
samples (see Materials and methods) the
vitreous preparation contained 31,600
c.p.m. and the corneal extract, 25,300
c.p.m. Each was digested with Pronase
and chromatographed on Sephadex G-25.
In the cornea (Fig. 4) most of the radioactivity was eluted in the first peak together with all of the chondroitin sulfate
and keratosulfate. Relatively little radioactivity was found in the ninhydrin-positive effluents. It should also be noted that
in the gel filtration chromatogram of the
corneal extract there was only one major
ninhydrin peak; it appeared in the elution
profile after the mucopolysaccharide peak.
Although some ninhydrin-positive materials
were also eluted in the void volume of the
column together with the mucopolysaccharides, they did not form a well-defined
peak and apparently were not related to
any of the components appearing in the
first effluents, i.e., the mucopolysaccha-
rides. In contrast, two distinct ninhydrin
peaks were present in the vitreous chromatogram (Fig. 5), the first one coinciding in elution position with the hyaluronic
acid and the second (major) one appearing in the later effluents. It was also apparent that in the vitreous only a small
fraction of the total radioactivity was found
in the first (hyaluronic acid) fractions,
whereas in the corneal digests most of the
radioactivity was eluted in this position.
The mucopolysaccharide fractions from
both the cornea and the vitreous were
hydrolyzed and chromatographed on Dowex 50. These analyses (Table V) showed
that both the glucosamine and the galactosamine isolated from the comeal digests
contained significant amounts of radioactivity, whereas the glucosamine isolated
from the vitreous contained only 35 c.p.m.
Hence, during the 4 hour period of infusion of radioactive glucose in five consecutive experiments only a minimal
amount of hyaluronic acid synthesis had
taken place in the vitreous. The main
purpose of studying the incorporation into
the corneal mucopolysaccharides was to
test the experimental technique itself. Had
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Investigative Ophthalmology
October 1969
530 Berman and Gombos
Table V. Distribution of radioactivity in the mucopolysaccharides of the cornea and the
vitreous after infusion of U-"C-glucose through the lateral long ciliary artery*
Neutra , sugar
Galactosamine
Glucosamine
Amount
Radioactivity
Amount
Radioactivity
Amount
Radioactivity
(c.p.m.)
(c.p.m.)
(c.p.m.)
Tissue
(»g)
864
550
284
530
546
680
Cornea
327
Vitreous
190
258
35
"After gel filtration of Pronase-digested vitreous and cornea (Figs. 4 and 5, respectively), the first peak, which contained
all of the mucopolysaccharides, was hydrolyzed and chromatographed on Dowex 50. The anthrone-positive material
(neutral sugar) was found in the effluent fractions not retained on the Dowex 50 column; the amino sugars were
eluted afterward with HC1.
no radioactivity been found in the corneal
mucopolysaccharides either, it would have
cast considerable doubt on the adequacy
of this in vivo technique. Notwithstanding
the differences in the mucopolysaccharide
content of the two tissues,* the amount of
radioactivity incorporated into amino sugars by the cornea was about 40 times
greater than in the vitreous.
Discussion
Incorporation of labeled glucose into
hyaluronic acid in vitro. These studies have
revealed that the utilization of U-14C-glucose for hyaluronic acid synthesis in the
vitreous can be demonstrated when relatively large amounts (50 ^ic) of labeled
glucose are present in the incubating medium. When small amounts (10 or 20 /xc)
are used, none of the radioactivity incorporated into the nondialyzable polymers is
present in the glucosamine. Although conversion to this amino sugar has been used
as an "indicator" of hyaluronic acid synthesis, it should be pointed out that 5
to 10 per cent of the glucosamine in these
preparations may have originated from
glycoproteins. Neglecting the contribution
from this source would not alter the interpretation of the findings reported here.
Considerably more radioactivity is incorporated into vitreous polymers using
50 /xg of labeled glucose but, even under
these conditions, the amount converted to
"The total content of hyaluronic acid in a single cat
vitreous is approximately 75 /ig30; the total mucopolysaccharide content of a single cat cornea varies from 400 to
600 /tg (Salitemik-Givant and Berman, unpublished observations).
glucosamine represented only 1 to 2 per
cent of the total radioactivity applied to
the Dowex 50 column (Table II). Moreover, longer periods of incubation did not
result in any greater synthesis of labeled
glucosamine (Table III).
Of all the factors investigated, only the
age of the animal appears to influence
the extent of hyaluronic acid synthesis.
Nearly six times more radioactivity was
found in the amino sugars isolated from
young (4-week-old) calves than from
adult (1- to 2-year-old) cattle. It should
be pointed out. however, that these experiments may not be strictly comparable
because twice as many vitreous-ciliary
body preparations were required from 4week-old calves than from adult animals
in order to obtain equivalent amounts of
uronic acid (Table III). It may therefore
be argued that, in the experiments with
4-week-old calf vitreous, the amount of
tissue or the number of cells7 actually incubated was at least twice as great as that
in the older animals. In spite of this, the
results of the present experiments are in
good agreement with previous chemical
studies22 which showed that net synthesis
of hyaluronic acid occurs mainly during
the early postnatal period of growth in the
cattle eye. The amount of radioactivity
found in the glucosamine isolated from
vitreous of older animals was so low that
its significance must be considered as
questionable.
Glucose is a major precursor of hyaluronic acid in other biological systems13
and, by analogy, it had been anticipated
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Volume 8
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U-"'C-glucose and vitreous polymers 531
that this would likewise hold true for the
vitreous. Enzymes capable of transferring
the labeled carbohydrate moieties of UDPglucuronic acid-14C3 and UDP-N-acetylgrucosamine-llC3r< to endogenous hyaluronic acid acceptors have been demonstrated in high-speed sediments of calf
vitreous. These findings do not necessarily
contradict the present investigations because they represent the final steps in the
synthesis of hyaluronic acid. Glucose is
converted to uridine nucleotide sugars by
enzymes found widely distributed in nature but to date only one of them, a
transferase that converts l-^'C-glucosamine
to labeled UDP-N-acetylglucosamine, has
been isolated from the vitreous.30 That others may also be present in this tissue
may be inferred from the observation that
after injection of labeled glucose into the
aqueous of an enucleated calf eye, some
radioactivity was found in alcohol-soluble
oligosaccharides present in the central
vitreous.30 Since whole eyes were used
in these experiments, the actual site of
conversion of glucose to nucleotide sugars
could not be established.
Fate of radioactive glucose in vitro. The
finding that only a small fraction of the
incorporated radioactivity was associated
with the glucosamine moiety of the hyaluronic acid prompted further investigations
on the metabolic fate of the remainder of
the glucose. Fractionation on DEAESephadex of vitreous preparations that had
been incubated with U-llC-glucose showed
that most of the radioactivity was in the
protein (and glycoprotein) fractions. Similarly, with Pronase-digested preparations
subjected to gel filtration, most of the
radioactivity was found in the low molecular weight peptide fractions eluted after
the hyaluronic acid. Studies on the distribution of radioactivity in the two principal
fractions obtained by gel filtration (Table
IV) showed that in this experiment 4 per
cent of the radioactivity recovered after
Dowex 50 chrornatography was associated
with the glucosamine. The remainder was
found in the neutral sugar and the nin-
hydrin-positive fractions. Although it was
not anticipated that such an extensive
conversion of glucose carbon to amino
acids would occur in the vitreous, a similar phenomenon has been observed in
brain81 and in surviving bone fragments
32, a3 w n e r e radioactive glucose is converted
more rapidly to amino acids than to other
monosaccharides.
The most convincing evidence supporting the view that, in the vitreous, glucose
participates mainly in the synthesis of proteins (and glycoproteins) and to a lesser
extent in the biosynthesis of hyaluronic
acid has come from studies using puromycin. A comparison of the gel filtration
patterns of Pronase-digested vitreous in
the presence (Fig. 3) and absence (Fig.
2) of puromycin reveals the marked inhibitory effect of this antibiotic. Further
examination of the distribution of the
radioactivity showed that, on an absolute
basis, the largest inhibition was in the
netutral sugar and ninhydrin-positive fractions. In contrast, conversion to glucosamine was only slightly decreased in the
presence of puromycin.
The inhibitory effect of rjuromycin on
protein synthesis at the ribosomal level
is well documented and it is now known
that this antibiotic also inhibits proteinpolysaccharide-synthesizing systems in embryonic chick cartilage,27' 2S in skin fibroblasts,-9'31 and in sheep mucosal scrapings.20 It is, therefore, reasonable to assume that the formation of the protein
or peptide backbone of the mucopolysaccharide not only precedes, but may actually be essential for, the subsequent incorporation of carbohydrate and sulfate
residues. On the other hand, the requirement of a preformed peptide core for
hyaluronic acid synthesis has not been
clearly established. The fact that incorporation of radioactive precursors into hyaluronic acid by skin fibroblasts was only
partially inhibited by puromycin29 suggests that its synthesis is less dependent
upon protein synthesis than that of other
polysaccharides or mucoproteins. In the
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Investigative Ophthalmology
October 1969
532 Berman and Gombos
vitreous the synthesis of hyaluronic acid
was only slightly (28 per cent) inhibited
by puromycin whereas that of proteins
and glycoproteins was depressed by approximately SO per cent. Therefore, the
over-all inhibition of 77 per cent in the
incorporation of labeled glucose into vitreous polymers reflects mainly an inhibition
of protein synthesis and, in this respect,
the puromycin-sensitive protein-synthesizing pathways in the vitreous appear to be
similar to those found in other tissues.
Glucose metabolism in vivo. It had
previously been established that after the
infusion of U-^'C-glucose through the lateral long ciliary artery of the cat considerable
amounts of radioactivity entered the vitreous.1'1 The use of this technique circumvents the problems inherent in the more
conventional routes of administration (intravenous or peritoneal injection) which,
because of the extensive dilution and rapid
metabolism of the administered glucose,
would not be expected to yield glucose of
high specific activity in the vitreous. Even
then, because of the low level of metabolic
activity anticipated in the cat vitreous, 5
separate experiments were performed and
the tissues (vitreous and cornea) were
pooled for subsequent analyses. It was
found that, although considerable radioactivity was present in the amino sugar
moieties of the comeal mucopolysaccharides, only borderline amounts (35 c.p.m.)
could be detected in the glucosamine isolated from the vitreous of 5 pooled cats'
eyes. This suggests that very little hyaluronic acid had been synthesized in vivo
during the 4 hour period of continuous
infusion of glucose through the lateral long
ciliary artery. Parenteral injection of U14
C-glucose also resulted in very little incorporation into vitreous hyaluronic acid
in the owl monkey.3> 37 Labeled glucosamine is a far more efficient precursor of
hyaluronic acid than glucose37 but, even
then, maximum incorporation does not
occur until 5 days after its administration.
The slow turnover of hyaluronic acid is
well known and it seems possible that the
4 hour period used in the present studies
was not sufficient to demonstrate biosynthesis.
Site of metabolic activity. It has been
proposed2"'1'!) that most, or possibly all, of
the metabolic activity of the vitreous resides in the cortical layer cells ("hyalocytes"). It was, therefore, surprising to
find that the central, acellular, portion of
the tissue (Preparation C) was nearly as
active as the surface layer (Preparations
A and B) in incorporating radioactive glucose into nondialyzable polymers. However, in view of the fact that soluble transferases, and possibly other enzymes, are
present in the acellular regions of calf
vitreous,30 the observation that Preparation C was so active may be explained
in terms of "extracellular enzymes."
The present investigations do not support the view that the ciliary body plays
a direct role in the production of vitreous
hyaluronic acid. In all experiments where
cortical layer vitreous was incubated either alone (Preparation B) or attached to
the ciliary body (Preparation A), there
was little difference in the total amount
of radioactivity incorporated, or in the
amount of labeled glucose converted to
glucosamine. If one were to suppose that
either hyaluronic acid, or some precursor
of it, is normally present in the ciliary
body, then it should be possible to detect
such a substance by chemical methods.
However, when the ciliary body was freed
of all adhering vitreous and extracted in
aqueous buffered media, no hyaluronic
acid could be found (Berman, unpublished observations). The Alcian bluestaining "mucoids" detected histochemically in the tissue11'12 must then be another
type of mucopolysaccharide and probably
not one that is related, either chemically
or metabolically, to the hyaluronic acid
of the vitreous.
The authors wish to thank Professor I. C.
Michaelson for his interest in this work and Mr.
Rami Shiloni for technical assistance.
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